Summary: The biochemical mechanism by which BoNTs enter and act upon nerve cells is not understood at the molecular level. A direct discovery in 1999 demonstrated that two of the seven BoNTs (Type A and E) that act on the same neuronal site have very different stabilities within the nerve cells. Type A toxin survives in cells beyond three months whereas type E is destroyed within a few days. The most likely explanation to account for this descrepancy is that a neuronal component, perhaps a protein, protects the type A toxin. The yeast-two hybrid technique will be used to screen protein-protein interactions between the type A botulinum neurotoxin and neuronal proteins. Proteins discovered to bind with the toxin will be isolated and tested in vitro and in whole-cell preparations. The role of phosphorylation in these interactions will be assessed because phosphorylation stabilizes the toxin's catalytic activity.